It is highly desired to develop new non-invasive imaging approach for detecting bacterial infection with improved specificity and spatial-temporal resolution. Although to date there are a number of molecular imaging approaches have been demonstrated for bacterial infection in the preclinical animal models, there is clearly a gap between the demand of clinically-compatible imaging methods and conventional tagging strategies, which often require the use of metallic or radioactive contrast agents. To address this gap, we are developing a ?non-labeled? (i.e., not radioactive, and not paramagnetic- or super-paramagnetic-) approach for detecting bacteria. In this approach bacterial cells are directly detected through their endogenous molecules using an innovative MRI technology called Chemical Exchange Saturation Transfer (CEST). The long-term goal of our research is to exploit this endogenous bacteria-specific CEST MRI signal to detect pathogenic bacteria and monitor the progress of bacterial infection. As initially demonstrated in solid tumors, we showed that CEST MRI could detect a therapeutic bacteria C. novyi-NT, a genetically modified bacteria strain currently in the Phase I clinical trial for treating solid tumors. Based on the preliminary data, we hypothesize that CEST MRI that detects the germination and proliferation of C. novyi-NT can be used as a non-invasive imaging biomarker for predicting the success of bacteriolytic therapy on glioma, a highly lethal brain tumor type. We plan to test our hypothesis through completing the following two specific aims: 1) To improve the specificity of bacterial CEST contrast using newly developed CEST technologies, and 2) To test and validate the optimized bacterial CEST MRI detection in the C. novyi-NT cancer therapy. We anticipate that accomplishing these aims will result in a highly translatable MRI technology specifically for bacterial detection, which could be used immediately as an imaging biomarker in the clinical trials of C.novyi-NT bacteriolytic therapy. More importantly, the knowledge gained from this project will enable a further expansion of using the proposed CEST MRI methods to the detection of many other types of bacteria, which will be enormously useful for the diagnosis and treatment monitoring of bacterial infection in deep organs, such as brain, which are currently difficult to detect.